The effects of amiloride, an ion channel blocker, on alamethicin pore

Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC

Biological and Environmental Phenomena at the Interface

The effects of amiloride, an ion channel blocker, on alamethicin pore formation in negatively charged, gold supported, phospholipid bilayers – a molecular view. Fatemeh Abbasi, ZhangFei Su, Julia Alvarez Malmagro, J. Jay Leitch, and Jacek Lipkowski Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00187 • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 25, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

amiloride changes alamethicin pore structure 254x190mm (96 x 96 DPI)

ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The effects of amiloride, an ion channel blocker, on alamethicin pore formation in negatively charged, gold supported, phospholipid bilayers – a molecular view. Fatemeh Abbasi, ZhangFei Su, Julia Alvarez-Malmagro, J. Jay Leitch and Jacek Lipkowski* Department of Chemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1

Abstract The effects of amiloride on the structure and conductivity of alamethicin ion pore formation within negatively charged, gold supported, DMPC/Egg-PG membranes were investigated with the help of electrochemical impedance spectroscopy (EIS), photon polarization modulation infrared reflection spectroscopy (PM-IRRAS) and atomic force microscopy (AFM). The EIS results indicate that ion conductivity across negatively charged phospholipid bilayers containing alamethicin is decreased by an order of magnitude when amiloride is introduced to the system. Despite the reduction in ion conductivity, the PM-IRRAS data shows that amiloride does not inhibit ion channel formation by alamethicin peptides. High resolution AFM images revealed that amiloride enlarges and distorts the shape of alamethicin ion pores when introduced to the system indicating that it is inserting itself into the mouth of the alamethicin pores. This effect is driven by electrostatic interactions between positively charged amiloride molecules and negative charge on the membrane.

1. Introduction Amiloride is a drug used to inhibit ions transport through channels in biological cells. Many amiloride analogs have been synthesized and are commercially available.1 It is widely understood that amiloride, a substituted pyrazinoylguanidine, and series of its analogs bind to alamethicin ion channels through electrostatic interactions or via molecular interactions between amiloride and aggregated alamethicin molecules reducing the ion channel conductivity.2,3 It has also been reported that peptide polycations, such as salmon protamine (MW = 4000 g/mol) and poly-L-lysine (MW = 100,000 g/mol), and positively charged proteins, such as streptavidin (MW 1 ACS Paragon Plus Environment

Page 2 of 26

Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

= 40,000 g/mol) are capable of blocking ion translocation through alamethicin channels.4,5 Amiloride is a small molecule (MW = 230 g/mol) making it an ideal candidate for obtaining a molecular level description of the interactions between amiloride and alamethicin and providing a better understanding ion channel inhibition. Alamethicin (Alm) is a short, 20 amino acid antimicrobial peptide isolated from fungus Trichoderma viride.6 Alamethicin adopts a helical secondary structure consisting of a 13 residue α-helix subunit connected to a 310-helix subunit by a kink formed by a proline residue at position14.7 Alamethicin has amphiphilic properties and binds to the surface of the lipid bilayer (S state) at low peptide/lipid (P/L) molar ratios or inserts into the bilayer (I state) at high P/L ratios.8 In the I state, alamethicin helices aggregate to form a barrel stave transmembrane pore creating a water channel that permits the transport of small ions across the bilayer.7,9,10 In a series of recent papers, we have employed electrochemical scanning tunneling microscopy (EC-STM)11 to image the pore formed by alamethicin in a lipid monolayer and atomic force microscopy (AFM)12,13 to obtain images of the pore in floating phospholipid bilayers. The insertion of the peptide into the bilayer was monitored with the help of photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and the pore conductivity was measured using electrochemical impedance spectroscopy (EIS).14,15 Independently, EIS has been used to study the voltage-gated formation of alamethicin channels in bilayers supported on mercury electrode surfaces by Guidelli and coworkers.16,17 Recently, Forbig et al. applied surface enhanced IR spectroscopy to study dynamics of alamethicin channel formation in a tethered bilayer.18 The objective of this work is to apply our experience with alamethicin channel formation in model biomimetic membranes to provide a molecular level description of the blocking action by amiloride. Previously, the blocking of alamethicin pores by amiloride was investigated by EIS.2 In this work, we will employ AFM and PM-IRRAS to demonstrate how amiloride affects the membrane and alamethicin pore structure to reduce ion channel conductivity. Our experiments will be performed using a floating bilayer lipid membrane (fBLM) composed of a mixture of negatively charged Egg-PG and zwitterionic DMPC phospholipids supported at a 1thio-β-D-glucose (β-Tg) modified gold (111) electrode described in details in ref.13 This work will contribute to the understanding of the molecular mechanism of the function of ion channel blockers. 2 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2. Experimental Section The experimental methods and procedures employed in this study were similar to that described in ref.13 To avoid repetition, a brief description of the procedure is described in the following section and a more detailed description has been reported in Section 1 of the Supporting Information (SI). Chemicals and Solutions. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and L-αphosphatidylglycerol sodium salt derived from chicken egg (Egg-PG) were purchased from Avanti Polar Lipids (99% pure; Alabaster, AL, US). Alamethicin (Alm), 1-thio-β-D-glucose (βTg) and amiloride hydrochloride hydrate (98%, pKa = 8.7) were purchased from Sigma-Aldrich (Oakville, ON, CA). The phospholipids were used as purchased without further purification. HPLC-grade chloroform purchased from Sigma-Aldrich (Oakville, ON, CA) was used to prepare vesicle solutions. Sodium fluoride powder (99% purity), purchased from Sigma-Aldrich (Oakville, ON, CA), was cleaned in a Jelight UV-ozone chamber (Irvine, CA, US) for 20 min to oxidize organic impurities prior to use. The NaF electrolyte solutions were prepared by dissolving the pre-cleaned powder in ultrapure water (18.2 MΩ cm) obtained from an EMD Millipore UV plus water system (Billerica, MA, US) to give final concentrations of 100 mM in the EIS and PM-IRRAS experiments and 1 mM in the AFM measurements.

Sample Preparation and Bilayer Formation. All glassware was cleaned in a hot mixed acid bath (1-part HNO3 : 3-parts H2SO4) for 60 min and then thoroughly rinsed and soaked in Milli-Q water for 3 – 4 h. The Teflon pieces for the cells were soaked in a Piranha solution (1-part H2O2 : 3-parts H2SO4) and then rinsed with Milli-Q ultrapure water. The AFM gold substrates were produced in an AXXIS co-sputtering deposition system (Kurt J. Lesker, Jefferson Hills, PA, US) by magnetron sputtering technique (Torus, 3" dia., DC). Titanium adhesion layers (3 – 5 nm thick) were first deposited onto clean glass microscope slides. Next, gold films of ~200 nm were deposited onto the titanium adhesion layers to create stable and highly uniform gold substrates. The gold coated glass slides were then annealed in a muffle furnace at 675 °C for 70 s to produce large, well-ordered gold crystallites. The gold slides

3 ACS Paragon Plus Environment

Page 4 of 26

Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

were then immersed into a 2 mM aqueous solution of β-Tg for 20 h to deposit a uniform selfassembled monolayer (SAM). Stock solutions of DMPC, Egg-PG and alamethicin were prepared by dissolving each powder in chloroform to give final concentrations of 4.54, 10 and 2 mg/ml, respectively. The appropriate concentration of each stock solution was then combined in a single test tube. The solvent was then slowly evaporated by vortexing the mixture under a stream of argon producing thin, dried films of either DMPC/Egg-PG (1:1 molar ratio) or DMPC/Egg-PG/Alm (9:9:2 molar ratio) and these test tubes were placed in a vacuum desiccator for at least 24 h prior to use to ensure that the chloroform was completely removed. The vesicles were then created in the test tubes by adding 1 ml of Milli-Q water to the dry lipid film to give a final concentration of 1 mg/ml. The mixture was sonicated for 30 min at 35 °C to ensure that vesicles, with the appropriate size and distribution, were formed in water. The final concentration of amiloride used in the experiments was 60 μM. AFM Measurements. Dynamic MAC mode AFM images were acquired with an Agilent Technologies 5500 Scanning Probe Microscope (Agilent N9621-13601 MAC III Mode controller, Santa Clara, CA, US) in a 1 mM NaF solution. All images were recorded at 21 ± 0.5˚C using type VII MAC cantilevers (Keysight Technologies, Kanata, ON, CA) with nominal spring constant of 0.14 N/m and resonance frequency of 8-10 kHz in solution. Data acquisition and analysis were carried out using PicoView 1.49 (Agilent Technologies, Mississauga, ON, CA) and Gwyddion v2.40 (Czech Metrology Institute, Brno, CZ) software, respectively. The force versus distance curve measurements were carried out in solution using V-shape silicon nitride cantilevers with spring constants of ~0.06 N m-1. A home-made EC-AFM cell was employed for potential dependent studies. A gold wire and an oxidized gold wire were used as counter electrode and reference electrode respectively. The potentials were applied using the potentiostat/galvanostat (HEKA PG590, Pfalz, DE). Electrochemical Instrumentation and Measurements. The electrochemical measurements were carried out in an all-glass three-electrode cell consisting of a gold (111) working electrode, a coiled platinum wire as the counter electrode and a saturated Ag/AgCl reference electrode (sat. 4 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

KCl, +197 mV vs. SHE). A single crystal gold (111) electrode with a surface area of 0.172 cm2, prepared in accordance to the procedure described in ref.20 was used as the working electrode for the electrochemical impedance spectroscopy (EIS). These measurements were performed using the Solartron SI 1287 electrochemical interface (Ametek Scientific Instruments, Oakridge, TN, US) and Solartron SI 1260 impedance/gain-phase analyzer (Ametek Scientific Instruments, Oakridge, TN, US). EIS spectra were collected in the frequency range from 10-2 to 103 Hz at different bias potentials ranging from 0.1 V to -0.4 V vs. Ag/AgCl electrode and ac amplitude of 0.005 V. Data processing of the EIS results was conducted using the ZView software (Scribner Associates Inc., Brno, CZ). PM-IRRAS Measurements. PM-IRRAS experiments were performed using a Thermo Nicolet Nexus 870 spectrometer equipped with an external tabletop optical mount (TOM) box. An electrochemical IR cell equipped with 1 in. CaF2 equilateral prism (BoXin, Changchun, CN) was used in the IR experiment. The prism was washed with methanol and pure water, and then cleaned in the UV ozone chamber for 15 min prior to mounting it on the electrochemical cell. The half-wave retardation of the photoelastic modulator (PEM) was set to 1600 cm-1 using a 60o angle of incidence for the amide I band region and 2900 cm-1 at an angle of 55o for the CH stretching region. Heavy water (D2O) was selected as solvent to avoid spectral overlap by the strong IR absorption bands of water (H2O). To ensure that the measurements had adequate signal-to-noise (S/N), 4000 spectral scans were collected and averaged with the instrumental resolution of 4 cm-1 for each potential ranging from 0.1 and –1.0 V vs. Ag/AgCl (sat.). The average and the difference signals from the detector were corrected for the PEM response functions as described in ref.21 A detailed description of procedures used to calculate tilt angles of the acyl chains of the lipids and alamethin helices are described in Section 1 of the Supporting Information.

3. Results and Discussion 3.1 Electrochemical characterization of amiloride on electrode supported phospholipid bilayers 3.1.1 Differential capacitance curves 5 ACS Paragon Plus Environment

Page 6 of 26

Page 7 of 26

The effect of amiloride on the stability of the fBLM was investigated by differential capacitance. Figure 1 shows the differential capacitance curves for the bare gold (111) electrode (curve1), electrode covered by a monolayer of β-Tg (curve 2), DMPC/Egg-PG/Alm bilayer (curve 3) and DMPC/Egg-PG/Alm bilayer with amiloride (curve 4).

70 60

1

-2

Capacitance / F cm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

50 40 30

2

20 3

10 0

4 -1.2

-0.8

-0.4

0.0

0.4

E / V vs. Ag AgCl

Figure 1. Differential capacitance curves of the film-free gold electrode (1-blue), β-Tg SAM modified gold electrode (2-black), floating DMPC/Egg-PG/Alm bilayer on the -Tg-modified gold electrode surfaces (3-green), floating DMPC/Egg-PG/Alm bilayer in the presence of 60 μM concentration of amiloride in the electrolyte (4-red) recorded at a scan rate of 5 mV s-1, frequency 25 Hz and 5 mV rms ac amplitude in a 0.1 M NaF electrolyte. The inset shows the structure of the amiloride molecule.

The data shows that amiloride causes an increase in the capacitance of the fBLM in the potential window between +0.2 and –0.6 V vs. Ag/AgCl, but significantly decreases the capacitance at potentials more positive than +0.2 V vs. Ag/AgCl. The increased capacitance of the fBLM in the absence of amiloride compared to the film covered electrode in the presence of amiloride at more positive potentials may correspond to the penetration of anions into defects within the bilayer. This suggests that amiloride is able to either suppress suppresses anion penetration or reduce the defect density at these positive potentials. The differential capacitance curves indicate that the 6 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 26

bilayers are detached (desorbed) from the electrode surface at potentials more negative than –0.8 V vs. Ag/AgCl. The detachment is triggered by desorption of β-Tg monolayer. It is interesting to note that the desorption (detachment) peaks are significantly reduced in the presence of amiloride. Since the amplitude of desorption peaks is controlled by kinetics, this behavior suggests that amiloride slows down the detachment of the fBLM. 3.1.2 Electrochemical impedance measurements Initially, the EIS measurements were performed on the DMPC/Egg-PG without alamethicin to check the impact of amiloride on the stability of the pure phospholipid (DMPC/Egg-PG) bilayer. The results are described in Section 2 of the Supporting Information, which show that amiloride has a negligible effect on the DMPC/Egg-PG bilayer in the absence of alamethicin. Figures 2 (a) and (b) show the total impedance and corresponding phase angles of the DMPC/Egg-PG/Alm bilayers in the absence and presence of amiloride at a potential +0.1 V vs. Ag/AgCl. There are minimal differences in the absolute values of the impedance but Figure 2 (b) shows that the phase angle values are larger at frequencies below 1 Hz in the presence of amiloride. Valincius et al.22 demonstrated that a decrease in the phase angle at low frequencies indicates presence of defects or pores in the bilayer. This implies that the increase in the phase angle observed in Figure 2 (b) by the addition of amiloride to the system, qualitatively indicates that amiloride is blocking the alamethicin pore. An equivalent circuit proposed by Valincius et al.22, shown in Figure 3 (a), was used to model the electrode interface and obtain numerical values of resistance and capacitance of the DMPC/Egg-PG/Alm bilayer in the absence and presence of amiloride. In this circuit, Rs and Rm are resistances associated with the electrolyte solution and phospholipid bilayer (membrane), CPEm and CPEsub represent constant phase elements of the membrane and the sub-membrane layer separating the bilayer from the gold electrode surface. The capacitance is represented by a CPE element to account inhomogeneities that may be present in the bilayer. The impedance of the constant phase element is described by the equation:

Z CPE 

1 Q( j )

(1)


Page 9 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

where Q is the constant phase element coefficient measured in μF cm-2 sα-1 and α is related to the frequency dispersion (if α = 1, Q, then it is equal to the capacitance; if α = 0, then ZCPE is frequency independent implying that the component is purely resistive). The equivalent circuit in Figure 3 (a) shows a good fit with the experimental EIS data for the bilayer in the absence of amiloride giving an αsub parameter of 0.5. This value is characteristic for Warburg impedance and indicates that the impedance of the sub-membrane layer is limited by diffusion across the alamethicin channels in the absence of the ion blocking molecule.

Figure 2. (a) Total impedance and (b) phase angle results obtained from the EIS measurements of the DMPC/Egg-PG/Alm (black points) and DMPC/Egg-PG/Alm/amiloride bilayers (red points) supported at the gold (111) electrode modified with a monolayer of β-Tg at E = +0.1 V vs. 8 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Ag/AgCl. Solid lines represent fits of the experimental data to the equivalent circuits shown in Figure 3. When this same equivalent circuit was used to fit the data for the DMPC/Egg-PG/Alm/amiloride film, the Zsub had a coefficient of α < 0.5 indicating that Zsub has predominantly resistive properties. This is illustrated by Figures S3 and S4 of the Supporting Information that show attempts to fit EIS data to Valincius’ model. For each case, αsub < 0.5 indicating that Zsub is dominated by the resistance of the sub-membrane layer. As a result, the equivalent circuit was simplified to the model shown in Figure 3 (b). However, in this case Rm represents the sum of the resistance of the membrane and the sub-membrane layer. In Figures 2 (a) and (b), the solid lines show fits of the experimental data to the equivalent circuit. The fits show excellent agreement with the experimental data producing reasonable numerical values for the elements of the equivalent circuits listed in Figure 3.

Figure 3. Equivalent circuits used to model the EIS data for the (a) DMPC/Egg-PG/Alm and (b) DMPC/Egg-PG/Alm/amiloride fBLMs

Table 1: Numerical values obtained for gold (111) electrodes covered with DMPC/Egg-PG/Alm bilayers in the absence and presence of 60 μM amiloride at E = +0.1 V vs. Ag/AgCl in 0.1 M NaF solution using the equivalent circuits described in Figure 3.


Page 10 of 26

Page 11 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

The resistance of the DMPC/Egg-PG/Alm bilayer increases by an order of magnitude from a value of 0.89 ± 0.08 MΩ cm2 to a value of 10.6 ± 3.1 MΩ cm2 when amiloride is added to the system indicating that amiloride is preventing ion translocation through the alamethicin pores. This result is consistent with the earlier studies by the Cornell group.2 The values of parameter α are close to unity indicating that the CPEm coefficients can be considered as a good measure of the membrane capacitance where the membrane capacitance in the absence of amiloride has a value of 9.4 ± 1.6 µF cm-2 and increases to 16.3 ± 2.3 µF cm-2 when amiloride is present. The membrane capacitance, C, is equal to the ratio of the permittivity, 𝜖, over the 𝜖

thickness, d, of the adsorbed bilayer (i.e. 𝐶 = 𝑑). The increase in the membrane capacitance indicates an increase of 𝜖 (membrane thinning is excluded on the basis of AFM measurements presented later) and suggests that polar amiloride molecule is inserted into the bilayer.


Langmuir

E- Epzfc / V -0.5

-0.4

-0.3

-0.2

-0.1

0.0

(a)

Rm / M cm

2

10

1

0.1

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

E / V vs. Ag/AgCl 35

-0.5

-0.4

E- Epzfc / V -0.3

-0.2

-1

30

-0.1

0.0

(b)

25

-2

Qm /F cm s

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 26

20 15 10 5 0

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

E / V vs. Ag/AgCl Figure 4. Variation of the (a) membrane resistance (Rm) and (b) constant phase element (Qm) of the DMPC/Egg-PG/Alm bilayers in 0.1 M NaF electrolyte with 60 μM amiloride (black rectangles) and without amiloride (red circles) as a function the applied potential. The blue triangles represent the data of DMPC/Egg-PG bilayers in 0.1 M NaF electrolyte without amiloride.


Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

The EIS measurements were also performed for potentials region from 0.1 V to –0.4 V vs. Ag/AgCl corresponding to a broad minimum on the capacitance curve in Figure 1. These measurements are described in Section 3 of the Supporting Information. The equivalent circuits in Figure 3 used to the EIS data provided numerical values of Rm and Qm illustrating the impact of the electrode potential on the membrane resistance and capacitance. The numerical data is presented in Table S2 of the Supporting information. Figure 4 (a) and (b) compare Rm and Qm values determined for three bilayers: DMPC/Egg-PG, DMPC/Egg-PG/Alm, and DMPC/EggPG/Alm/amiloride. The values of Rm in Figure 4 (a) are plotted on a logarithmic scale because the membrane resistances of the DMPC/Egg-PG/Alm bilayers are an order of magnitude lower than of the two other bilayers. These results show that the addition of amiloride blocks ion translocation through the channels in alamethicin-incorporated membranes restoring the membrane values observed for DMPC/Egg-PG bilayers without alamethicin. It should be emphasized that EIS data for both the DMPC/Egg-PG and DMPC/Egg-PG/Alm were fitted to the model presented in Figure 3 (b). To understand the molecular interactions that exist between amiloride and the alamethicin ion channels, PM-IRRAS and AFM data has been presented in Sections 3.2 and 3.3. Similar potential induced changes of Rm are observed for the pure DMPC/Egg-PG and DMPC/Egg-PG/Alm/amiloride bilayers. For the DMPC/Egg-PG bilayer, the decrease of Rm with negative potential indicates formation of defects (electroporation). A similar trend is observed in the presence of amiloride. Apparently, the presence of amiloride slightly increases the number of defects in the bilayer formed by electroporation. Figure 4 (b) compares Qm for the three bilayers. The Qm values for the DMPC/Egg-PG bilayer with and without alamethicin are comparable. The values for the DMPC/Egg-PG/Alm bilayer with amiloride are apparently higher than the other two systems. As mentioned earlier, this increase indicates that amiloride, which is a polar molecule, is inserted into the bilayer. In conclusion, EIS measurements provide information about the effect of amiloride on electrochemical properties of the membrane, however, structural information cannot be ascertained. To gain molecular level details about the interactions between amiloride and the model phospholipid bilayers, PM-IRRAS and AFM were employed.

3.2 PM-IRRAS measurements 12 ACS Paragon Plus Environment

Langmuir

At a concentration of 60 μM, the IR bands of amiloride present in the electrolyte solution residing within the thin layer cavity between the IR window and gold electrode were below the detection limits, which is illustrated in Figure S5 of the Supporting Information. Therefore, the IR spectra only consisted of IR absorption bands pertaining to the phospholipids and alamethicin peptides. The PM-IRRAS spectra of the CH stretching and amide I regions of the DMPC/EggPG/Alm bilayer with amiloride are shown in Section 4 of the Supporting Information. The spectra for CH stretching region (Figure S6) were deconvoluted and the integrated intensities of the symmetric and asymmetric methylene stretching bands were then used to calculate average tilt angle of trans fragments of the acyl chains (γchain) of the two lipids with respect to surface normal according to Equations S2 and S3. The red points in Figure 5 plot the γchain values of the DMPC/Egg-PG/Alm bilayer in the presence of amiloride as a function of the electrode potential. For comparison, the black squares

50

Tilt angle / degree

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 26

40 30 20 10 0 -1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

E / V vs Ag/AgCl

Figure 5. Dependence of the average tilt angle of the acyl chains (γchain) of the DMPC/EggPG/Alm bilayers floating on the β-Tg-modified gold (111) electrode in absence (black squares) and presence (red circles) of amiloride as a function of applied potential.

present the γchain values of the DMPC/Egg-PG/Alm bilayer without amiloride taken from ref 13.The

addition of amiloride causes an apparent increase of the tilt angle indicating that the acyl


Page 15 of 26

chains are more disordered. In the absence of amiloride, the changes of the tilt angle with potential correlate well with the shape of the differential capacitance curve shown in Figure 1. When amiloride is present, the changes of γchain with potential are smaller. This also is a manifestation of a more disordered bilayer state. The amide I bands (Figure S7 (a)) were used to calculate the average tilt angle of the primary axes of the α-helix and 310-helix of alamethicin peptides. The IR bands were deconvoluted (Figure S7 (b)) and the integrated intensities of the α-helix and 310-helix were calculated. The integrated intensities were then used to calculate the average values of the angles between the long axes of the two helices using the procedure described in Section 1 of the Supporting Information. The calculated tilt angles are plotted in Figure S8 of the Supporting Information and presented in in Figures 6 (a) and (b) where the red points plot the tilt angles of the two helices in the DMPC/Egg-PG/Alm bilayer when amiloride is present. For comparison, black squares in these 50

Tilt angle / degree

40 30 20 10

(a) 0 80 70

Tilt angle / degree

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

60 50 40 30 20

(b)

10 -1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

E / V vs. Ag/AgCl

Figure 6. Comparison of the tilt angles of the primary axes of the (a) α-helix and (b) 310-helix of alamethicin molecules embedded into the DMPC/Egg-PG/Alm bilayers floating on the -Tgmodified gold (111) electrode in absence (black squares) and presence (red circles) of amiloride as a function of applied potential in a 0.1 M NaF electrolyte solution.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

figures represent the angle of helices in the bilayer without amiloride taken from ref.13 Surprisingly, the two data sets are very similar. The differences are almost within experimental errors. This similarity indicates that amiloride has a negligible effect on the insertion of alamethicin molecules into the floating bilayer. The potential dependent changes of the tilt angles were explained in ref.13 Briefly, the potential of zero free charge (Epzfc) of the gold electrode with the DMPC/Egg-PG/Alm bilayer is ~0.2 V vs. Ag/AgCl.13 By moving the potential in the negative direction, the tilt angles decrease until a minimum is reached at ~–0.3 V vs. Ag/AgCl. This potential corresponds to a minimum on the differential capacitance curve presented in Figure 1. At more negative potentials, the tilt angles increase. This behavior correlates well with the increase of the electrode capacitance. Neutron reflectivity23 and SEIRAS24 experiments demonstrated that electroporation and electrodewetting of the bilayer from the gold electrode surface take place at these negative potentials. The differential capacitance curves in Figure 1 show that the membrane is detached from the gold surface at E < –0.8 V vs. Ag/AgCl. Here, the potential drop between the metal and electrolyte takes place in a thin layer of solution below the membrane, so that the membrane no longer experiences the potential applied to the gold electrode. Consequently, the tilt angles of the two helices return to the value observed at Epzfc. The PM-IRRAS data demonstrate that the insertion of alamethicin into the fBLM is potential driven. In summary, the PM-IRRAS measurements show that amiloride has a disordering effect on the membrane lipids but has no effect on the insertion of the alamethicin molecules. The behavior of alamethicin in the DMPC/Egg-PG bilayer, which is negatively charged, is different than in the DPhPC bilayer described previously in ref.15 The comparison of the PMIRRAS results of these two systems is discussed in Figure S9 of Supporting Information. At the negatively charged membrane, the studies were performed always at negative values of (E Epzfc) and the helices were less tilted by about 10o than the bilayer formed by zwitterionic lipids at the positive end of potentials. The lower values of the tilt angles indicate stronger insertion of alamethicin and stronger pore formation (i.e lower resistivity; see Table S3 of Supporting Information) in the negatively charged membrane. The amide I band is predominantly determined by collective vibrations of the C=O group of the amide bands. This IR band is very sensitive to the dipole-dipole coupling between 15 ACS Paragon Plus Environment

Page 16 of 26

Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

transition dipoles of vibrating oscillators. Therefore, a change in the helix structure leaves a signature in the position of the amide I band. Table 2 lists positions of amide I bands of -helix and 310-helix in DMPC/Egg-PG/Alm bilayers with and without amiloride. The positions of the

-helix of the amide I bands are within experimental error similar in solutions without and with amiloride. In contrast positions of 310-helix bands are shifted towards high wavenumbers by 2-3 cm-1 in the presence of amiloride. These shifts indicate a weakening of the dipole-dipole coupling in the 310-helix band. Alamethicin inserts into the bilayer with the hydrophobic Nterminus (-helix) embedded into the membrane and the hydrophilic C-terminus (310-helix) forming the mouth of the channel.16,17 The data listed in Table 2 indicate that amiloride does not interact with the -helix inserted deeply into the membrane but affects the structure of 310-helix helix located at the entrance to the channel blocking the channel. Table 2 Peak center (cm-1) of -helix and 310-helix bands of Alm in DMPC/Egg-PG/Alm bilayers in 0.1 M NaF electrolyte with and without amiloride at different potentials

DMPC/Egg-PG/Alm Potential / V 0.1 0.0 -0.1 -0.2 -0.3 -0.4

310-helix 1633 ± 1 1633 ± 1 1633 ± 1 1633 ± 1 1633 ± 1 1633 ± 1

-helix 1657 ± 1 1657 ± 1 1656 ± 1 1656 ± 1 1656 ± 1 1655 ± 1

DMPC/EggPG/Alm/amiloride 310-helix -helix 1636 ± 1 1657 ± 1 1635 ± 1 1657 ± 1 1635 ± 1 1658 ± 1 1636 ± 1 1658 ± 1 1634 ± 1 1657 ± 1 1634 ± 1 1657 ± 1

3.3 AFM studies Preliminary AFM experiments were performed with the DMPC/Egg-PG bilayer without alamethicin. The topography and phase images are shown in Figure S10 of the Supporting Information. In the presence of amiloride, the bilayer becomes smoother and more homogenous, however these changes are small. The force spectroscopy data for the DMPC/Egg-PG in the absence of alamethicin is also presented in Figure S11 of the Supporting Information.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(a)

(b)

Figure 7: AFM topography images of: (a) DMPC/Egg-PG/Alm (10 mol%) bilayer deposited on the β-Tg-modified gold (111) substrate by vesicle fusion and the (b) DMPC/Egg-PG/Alm membrane 3 h after the addition of amiloride (60 µM). The size of all four images is 50 × 50 nm2. The images were recorded at E = 0.1 V vs. Ag/AgCl.

These results demonstrate that amiloride has a minimal effect on the thickness of the bilayer. The magnitude of the penetration force for the DMPC/Egg-PG bilayer is slightly reduced when amiloride is added to the solution and the peak width becomes narrower (i.e. slightly more uniform). The small changes in the force curve results suggest that amiloride has a negligible effect on the nanomechanical properties of the bilayer in the absence of alamethicin. Figure 7 compares the topography images of the gold (111) electrode surface with DMPC/Egg-PG/Alm bilayer without amiloride (Figures 7 (a)) to the image recorded in the presence of amiloride (Figures 7 (b)). Figure S12 of Supporting Information shows the complementary amplitude images. The images show that the DMPC/Egg-PG/Alm bilayers are homogenous (i.e. no phase separation). After the addition of amiloride, the bilayer appears tob be much smoother. To compare nanomechanical properties of the two bilayers, Figure 8 shows the force distance curves of the DMPC/Egg-PG/Alm bilayers in the absence (Figures 8 (a)) and presence (Figure 8 (d)) of amiloride. Both sets of force curves show a discontinuity characteristic for the penetration of the bilayer by the tip, which is used to estimate the bilayer thickness. For each bilayer type, 120 force distance curves were measured at different positions on the gold


Page 18 of 26

Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(111) surface and histograms of the penetration distance (Figures 8 (b) and (e)) and penetration force (Figures 8 (c) and (f)) were constructed.

Figure 8: Typical force-distance curves of DMPC/Egg-PG/Alm bilayers in the (a) absence and (d) presence of amiloride deposited on a β-Tg-modified gold substrate. Histograms of the film thicknesses determined from the jump-in widths on the force-distance curves and corresponding penetration forces of the (b, c) DMPC/egg-PG/Alm and (e, f) DMPC/Egg-PG/Alm/amiloride fBLMs measured in a 1 mM NaF electrolyte at 20 ± 1 °C.

The Gaussian distributions applied to histograms in Figure 8 (b) and (e) give average jump-in distances of 4.7 ± 0.7 nm for DMPC/Egg-PG/Alm and 4.5 ± 0.7 nm after incubating with amiloride for 3 h. After applying the Hertzian correction to the force-distance data, the corrected thickness values are 5.6 ± 0.7 nm and 5.4 ± 0.7 nm for the bilayer in the absence and presence of amiloride, respectively. Apparently, the presence of amiloride has negligible effect on the bilayer thickness even when alamethicin is incorporated into the film. The penetration forces are also comparable. In the absence of amiloride, the average penetration force is equal to 0.32 ± 0.1 nN. In the presence of amiloride, the force distribution is narrower and appears bimodal in shape suggesting that amiloride is producing small inhomogeneities into the film that are not visible in the topography and phase images. When fitting these two peaks with two Gaussian distributions, the peak maximum of the lower penetration force distribution occurs at 0.22 ± 0.07 nN and while the higher penetration force peak attains a maximum value at ~0.36 ± 0.07 nN. In summary, the 18 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

differences between nanomechanical properties of the bilayer in the absence and presence of amiloride are small. 2.3 Potential dependent changes

Figure 9: AFM topography images of images of the DMPC/Egg-PG/Alm bilayer after incubating with 60 μM solution of amiloride for 3 h at (a) +0.1 V, (b) –0.1 V, (c) –0.3 V and (d) –0.4 V vs. Ag/AgCl. The scan size of the images is 50 × 50 nm. Figure 9 shows the AFM topography images of the DMPC/Egg-PG/Alm/amiloride bilayer when the potential is varied from + 0.1 to –0.4 V vs. Ag/AgCl. The topography AFM images show small changes in the bilayer structure with minor changes in the overall surface roughness. Unlike the previous images of the DMPC/Egg-PG/Alm bilayer, which show signs of swelling at negative potentials preceding the desorption of the bilayer,13 there are no signs of swelling or significant changes in the film morphology at negative potentials when amiloride is introduced to the DMPC/Egg-PG/Alm bilayer. The lack of bilayer swelling suggests that 19 ACS Paragon Plus Environment

Page 20 of 26

Page 21 of 26

amiloride inhibits water penetration into the film and prevents the translocation of electrolyte ions across the bilayer to the subphase region. However, the presence of the alamethicin pores could be seen in images of Figure 9. A comparison of images in Figures 7 (a) and 7 (c) shows that the pores are much larger when amiloride is present in the solution. Furthermore, the shape of the pores is quite regular, and the pores appear to be quite uniform in size. In contrast, the addition of amiloride to the DMPC/Egg-PG/Alm bilayer causes the alamethicin pores to become more distorted and the pore sizes appear more heterogeneous. (b)

0.1 V

0.3 0.2

0.0

0.0

2

4

6

8 10 12 14

0.3 0.2

0.0

0

2

4

6

8 10 12 14

0.3 0.2 0.1

0 2 4 6 8 10 12 14 Distance / nm

Distance / nm

5

-0.4 V

0.4

0.1

Distance / nm

Average pore diameter / nm

0.5

0.4

0.2

0.1

-0.3 V

0.5

0.3

0.1

0

-0.1 V

0.4 Height / nm

0.4

0.5

Height / nm

0.5

(d)

(c)

Height / nm

(a)

Height / nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

0.0

0 2 4 6 8 10 12 14 Distance / nm

(e)

4

3

2

1 0.2

0.1

0.0

-0.1

-0.2

-0.3

-0.4

Potential / V vs Ag/AgCl

Figure 10. High resolution AFM images (scan size of 20 × 20 nm) of DMPC/Egg-PG/Alm bilayers in the presence of amiloride obtained from the regions denoted by the squares in Figure 9 (a), (b), (c) and (d). The cross sections shown below each image were taken along the lines marked in the images shown above. (e) The average pore diameter determined for the DMPC/Egg-PG/Alm bilayer without (black points) and with the presence of amiloride (red points) as a function of the applied potential.

To analyze this effect in greater detail, Figures 10 (a), (b), (c), and (d) show zoomed in areas of images marked with frames in Figure 9. The cross-sectional profiles along the lines marked in images Figure 10 (a), (b), (c) and (d) are plotted below of each image. In addition, 20 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 10 (e) plots average pore diameters as a function of applied potential for bilayers without and with amiloride and the corresponding standard deviation. The results show that the average pore size is indeed higher in the presence of amiloride. Furthermore, the standard deviation associated with each pore dimension is also much larger, which reflects the distortion of the alamethicin pore shape by the interaction with amiloride. It should be noted that the pore dimensions could be only determined for well-shaped pores in the presence of amiloride suggesting that the pores distortion data in Figure 10 (e) is rather conservative and only accounts for alamethicin pores that are not significantly distorted by amiloride molecules. In conclusion, the AFM images provide direct information that the reduction in pore conductivity results from the blockage of alamethicin pore by amiloride and involves direct interactions between this channel blocker with the alamethicin pore, and not with individual peptides.

Conclusions EIS, PM-IRRAS and AFM were applied to investigate the effect of amiloride (an ion channel blocker) on the pore formation by alamethicin in negatively charged DMPC/Egg-PG bilayer floating at a gold (111) electrode surface. The results show that amiloride introduces some disorder of the acyl chains in the lipids, however, its overall effect on the bilayer thickness and nanomechanical properties is small. Amiloride molecules do not prevent the insertion and pore formation by alamethicin peptides in the phospholipid bilayer even though the conductivity of the membrane decreases by an order of magnitude when amiloride is present. High resolution AFM images demonstrate that this effect can be correlated to an increase in the alamethicin pore diameter and that amiloride causes a distortion in the size of the ion channels implying that amiloride is inserted into the pores. This is confirmed by an increase in the frequency of the 310helix band center indicating that amiloride is present at the surface of the alamethicin pore. It should be noted that the pKa of amiloride is 8.7 and our investigations were performed in a 0.1 M NaF solution, which has a pH of 8.1. In this electrolyte solution, the amiloride molecules are predominantly positively charged, therefore, blocking of the alamethicin channels can be explained by electrostatic attraction of positively charged amiloride molecules to the negatively charged phospholipid membrane. As a result, the amiloride molecules would reside at the membrane surface allowing them to interact with the alamethicin peptides at the mouth of the pore resulting in channel distortion and ion transport inhibition. 21 ACS Paragon Plus Environment

Page 22 of 26

Page 23 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Supporting Information 1. Detailed description of experimental procedures; 2. Effect of amiloride on DMPC/Egg-PG bilayers from EIS studies; 3. Effect of amiloride on DMPC/Egg-PG/Alm bilayers from EIS measurements; 4. PM-IRRAS spectra of the CH stretching vibrations of the phospholipid tails and amide I vibrations for the alamethicin peptides; 5. Atomic force microscopy studies of the effect of amiloride on DMPC/Egg-PG bilayers without alamethicin

Acknowledgement This work was supported by a Discovery grant from Natural Sciences and Engineering Council of Canada RG-03958.

References (1)

Kleyman, T. R.; Cragoe, E. J. Amiloride and Its Analogs as Tools in the Study of Ion Transport. J. Membr. Biol. 1988, 105 (1), 1–21. https://doi.org/10.1007/BF01871102.

(2)

Yin, P.; Burns, C. J.; Osman, P. D. J.; Cornell, B. A. A Tethered Bilayer Sensor Containing Alamethicin Channels and Its Detection of Amiloride Based Inhibitors. Biosens. Bioelectron. 2003, 18 (4), 389–397. https://doi.org/10.1016/S09565663(02)00160-4.

(3)

Yin, P. Tethered Bilayer Membrane Sensors with Small Transmembrane Peptide Ion Channels - Recent Developments, Future Research and Potential Applications. Adv. Planar Lipid Bilayers Liposomes 2005, 2 (C), 49–76. https://doi.org/10.1016/S15544516(05)02002-8.

(4)

Futaki, S.; Youjun, Z.; Sugiura, Y. Detecting a Tag on a Channel Opening: Blockage of the Biotinylated Channels by Streptavidin. Tetrahedron Lett. 2001, 42 (8), 1563–1565. https://doi.org/10.1016/S0040-4039(00)02287-5.

(5)

Rink, T.; Bartel, H.; Jung, G.; Bannwarth, W.; Boheim, G. Effects of Polycations on Ion Channels Formed by Neutral and Negatively Charged Alamethicins. Eur. Biophys. J. 22 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1994, 23 (3), 155–165. https://doi.org/10.1007/BF01007607. (6)

Leitgeb, B.; Szekeres, A.; Manczinger, L.; Vágvölgyi, C.; Kredics, L. The History of Alamethicin: A Review of the Most Extensively Studied Peptaibol. Chem. Biodivers. 2007, 4 (6), 1027–1051. https://doi.org/10.1002/cbdv.200790095.

(7)

Fox, R. O.; Richards, F. M. A Voltage-Gated Ion Channel Model Inferred from the Crystal Structure of Alamethicin at 1.5-A Resolution. Nature 1982, 300 (5890), 325–330. https://doi.org/10.2210/pdb1amt/pdb.

(8)

He, K.; Ludtke, S. J.; Heller, W. T.; Huang, H. W. Mechanism of Alamethicin Insertion into Lipid Bilayers. Biophys. J. 1996, 71 (5), 2669–2679. https://doi.org/10.1016/S00063495(96)79458-4.

(9)

Sansom, M. S. Alamethicin and Related Peptaibols--Model Ion Channels. Eur. Biophys. J. 1993, 22 (2), 105–124. https://doi.org/10.1007/BF00196915.

(10)

Mathew, M. K.; Balaram, P. A Helix Dipole Model for Alamethicin and Related Transmembrane Channels. FEBS Lett. 1983, 157 (1), 1–5. https://doi.org/10.1016/00145793(83)81105-3.

(11)

Pieta, P.; Mirza, J.; Lipkowski, J.; Murray, R. W. Direct Visualization of the Alamethicin Pore Formed in a Planar Phospholipid Matrix. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (52), 21223–21227. https://doi.org/10.1073/pnas.1201559110.

(12)

Abbasi, F.; Leitch, J. J.; Su, Z. F.; Szymanski, G.; Lipkowski, J. Direct Visualization of Alamethicin Ion Pores Formed in a Floating Phospholipid Membrane Supported on a Gold Electrode Surface. Electrochim. Acta 2018, 267, 195–205. https://doi.org/10.1016/j.electacta.2018.02.057.

(13)

Abbasi, F.; Alvarez-Malmagro, J.; Su, Z.; Leitch, J. J.; Lipkowski, J. Pore Forming Properties of Alamethicin in Negatively Charged Floating Bilayer Lipid Membranes Supported on Gold Electrodes. Langmuir 2018, 34 (45), 13754–13765. https://doi.org/10.1021/acs.langmuir.8b02554.

(14)

Su, Z.; Leitch, J. J.; Abbasi, F.; Faragher, R. J.; Schwan, A. L. EIS and PM-IRRAS Studies of Alamethicin Ion Channels in a Tethered Lipid. J. Electroanal. Chem. 2018,812,213-220 . https://doi.org/10.1016/j.jelechem.2017.12.039.

(15)

Su, Z.; Shodiev, M.; Jay Leitch, J.; Abbasi, F.; Lipkowski, J. In Situ Electrochemical and PM-IRRAS Studies of Alamethicin Ion Channel Formation in Model Phospholipid 23 ACS Paragon Plus Environment

Page 24 of 26

Page 25 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Bilayers. J. Electroanal. Chem. 2018,819,251-259. https://doi.org/10.1016/j.jelechem.2017.10.042. (16)

Becucci, L.; Guidelli, R.; Peggion, C.; Toniolo, C.; Moncelli, M. R. Incorporation of Channel-Forming Peptides in a Hg-Supported Lipid Bilayer. J. Electroanal. Chem. 2005, 576 (1), 121–128. https://doi.org/10.1016/j.jelechem.2004.09.032.

(17)

Guidelli, R.; Becucci, L. Mechanism of Voltage-Gated Channel Formation in Lipid Membranes. Biochim. Biophys. Acta - Biomembr. 2016, 1858 (4), 748–755. https://doi.org/10.1016/j.bbamem.2015.12.035.

(18)

Forbrig, E.; Staffa, J. K.; Salewski, J.; Mroginski, M. A.; Hildebrandt, P.; Kozuch, J. Monitoring the Orientational Changes of Alamethicin during Incorporation into Bilayer Lipid Membranes. Langmuir 2018, 34 (6), 2373–2385. https://doi.org/10.1021/acs.langmuir.7b04265.

(19)

https://avantilipids.com/product/841138

(20)

Richer, J. Measurement of Physical Adsorption of Neutral Organic Species at Solid Electrodes. J. Electrochem. Soc. 1986, 133 (1), 121. https://doi.org/10.1149/1.2108505.

(21)

Zamlynny, V.; Lipkowski, J. Quantitative SNIFTIRS and PM IRRAS of organic molecules at electrode surfaces, In Alkire, R.; Kolb, D. M.; Lipkowski, J.; Ross, P.N. Ed.; Diffraction and Spectroscopic Methods in Electrochemistry, Weinheim, Germany, 2006, Vol. 9, pp. 315-376. https://doi.org/10.1002/9783527616817.ch9.

(22)

Valincius, G.; Meskauskas, T.; Ivanauskas, F. Electrochemical Impedance Spectroscopy of Tethered Bilayer Membranes. Langmuir 2012, 28 (1), 977–990. https://doi.org/10.1021/la204054g.

(23)

Burgess, I.; Li, M.; Horswell, S. L.; Szymanski, G.; Lipkowski, J.; Majewski, J.; Satija, S. Electric Field-Driven Transformations of a Supported Model Biological Membrane—An Electrochemical and Neutron Reflectivity Study. Biophys. J. 2004, 86 (3), 1763–1776. https://doi.org/10.1016/S0006-3495(04)74244-7.

(24)

Uchida, T.; Osawa, M.; Lipkowski, J. SEIRAS Studies of Water Structure at the Gold Electrode Surface in the Presence of Supported Lipid Bilayer Dedicated to Professor Kingo Itaya on the Occasion of His 65th Birthday and in Recognition of His Seminal Contributions to Electrochemistry. J. Electroanal. Chem. 2014, 716, 112–119. 24 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

https://doi.org/10.1016/j.jelechem.2013.10.015. Table of contents graphics


Page 26 of 26

The effects of amiloride, an ion channel blocker, on alamethicin pore

Recommend Documents